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| TASK | WhoDunnit | Observed |
|---|
| Split cells | AG, CR, LA, SS | AW |
| Seed plates | AG, CR, LA, SS | AW |
| Transfection | AG, LA | AC, SS |
| FACS Prep | AG, LA | CR |
| Imaging (FloCyto) | BT | AG, AW, CR, JX, LA |
Practice Transfections:
CONTROLS
- -ve/-ve: no actions performed on cells
- -ve: perform protocol, excluding DNA, or with dummy DNA
- +ve: tri-color control (perform experiments with YFP, BFP, mKATE separately, and one with all 3: allows for comparison of color/intensity when imaging)
PRACTICE #1: Fluorescent protein under constitutive promoter
- 6/23: Seed cells
- 6/24: Transfect hEF1a:eYFP (472 ng/uL) into HEK cells
- 6/26 (4.30pm): Flow Cytometer
- Determine optimal Lipofectamine LTX volume (24 well)
- Determine transfection efficiency (quantitatively)
RESULT: Decent transfection efficiency (~60%). Move on to Practice #2.
PRACTICE #2: Co-transfection (2 fluorescent proteins under constitutive promoter)
ATTEMPT #1
- 7/3: Seed cells
- 7/4: Transfect hEF1a:eYFP (472 ng/uL) and hEF1a:tagBFP (1176 ng/uL) into HEK cells
- 7/6 (1pm): Flow Cytometer
- DNA Dilution: 10uL hEF1a:eYFP; 5uL hEF1a:tagBFP
- (Y+B)x: add 1:1 (25uL hEF1a:eYFP/tagBFP-lipid complex)
| Y1 | (Y+B)1 | ////// | ////// | ////// | -ve control- dummy DNA
- with Lipofectamine
|
| Y2 | (Y+B)2 | ////// | ////// | ////// | -ve control- dummy DNA
- with Lipofectamine
|
| B1 | (Y+B)3 | ////// | ////// | ////// | -ve/-ve control |
| B2 | (Y+B)4 | ////// | ////// | ////// | -ve/-ve control |
RESULT: Extremely low transfection efficiency (<10%). Repeat to improve co-transfection results.
ATTEMPT #2
- 7/8: Seed cells
- 7/9: Transfect hEF1a:eYFP (___ ng/uL) and hEF1a:tagBFP (___ ng/uL) into HEK cells
- 7/11 (_pm): Flow Cytometer
- DNA Dilution: __uL hEF1a:eYFP; __uL hEF1a:tagBFP
- (Y+B)x: add 1:1 (25uL hEF1a:eYFP/tagBFP-lipid complex)
| Y1 | (Y+B)1 | ////// | ////// | ////// | -ve control- dummy DNA
- with Lipofectamine
|
| Y2 | (Y+B)2 | ////// | ////// | ////// | -ve control- dummy DNA
- with Lipofectamine
|
| B1 | (Y+B)3 | ////// | ////// | ////// | -ve/-ve control |
| B2 | (Y+B)4 | ////// | ////// | ////// | -ve/-ve control |
RESULT:
PRACTICE #3: Fluorescent protein under inducible promoter
- 7/_: Seed cells
- 7/_: Transfect TRE:mKate, hEF1a:rtTA and hEF1a:eYFP (transfection marker) into HEK cells
- 7/_: Add DOX
- 7/_ (__pm): Flow Cytometer
- DNA Dilution: __uL hEF1a:eYFP; __uL hEF1a:rtTA; __uL TRE:mKate
- (D=x): add _:_:_
- __uL hEF1a:eYFP-lipid complex
- __uL hEF1a:rtTA-lipid complex
- __uL TRE:mKate-lipid complex
| +ve control | +ve control | -ve control- dummy DNA
- with Lipofectamine
| -ve control- dummy DNA
- with Lipofectamine
| -ve/-ve control | -ve/-ve control |
| ////// | ////// | ////// | ////// | ////// | ////// |
(D=0)1- no DOX
- hEF1a:eYFP
- hEF1a:rtTA
- TRE:mKate
| (D=0)2
- no DOX
- hEF1a:eYFP
- hEF1a:rtTA
- TRE:mKate
| D=0.2- 0.2uM DOX
- hEF1a:eYFP
- hEF1a:rtTA
- TRE:mKate
| D=1- 1uM DOX
- hEF1a:eYFP
- hEF1a:rtTA
- TRE:mKate
| D=1.5- 1.5uM DOX
- hEF1a:eYFP
- hEF1a:rtTA
- TRE:mKate
| D=3- 3uM DOX
- hEF1a:eYFP
- hEF1a:rtTA
- TRE:mKate
|
D=4- 4uM DOX
- hEF1a:eYFP
- hEF1a:rtTA
- TRE:mKate
| D=6
- 6uM DOX
- hEF1a:eYFP
- hEF1a:rtTA
- TRE:mKate
| D=7.5- 7.5uM DOX
- hEF1a:eYFP
- hEF1a:rtTA
- TRE:mKate
| D=10- 10uM DOX
- hEF1a:eYFP
- hEF1a:rtTA
- TRE:mKate
| D=17- 17uM DOX
- hEF1a:eYFP
- hEF1a:rtTA
- TRE:mKate
| D=30- 30uM DOX
- hEF1a:eYFP
- hEF1a:rtTA
- TRE:mKate
|
RESULTS:
PRACTICE #4**: Co-transfection (2 fluorescent proteins under constitutive & inducible promoters)
- 7/_: Seed cells
- 7/_: Transfect hEF1a:eYFP, TRE:mKate and hEF1a:rtTA** into HEK cells
- 7/_: Add DOX
- 7/_ (__pm): Flow Cytometer
